Systemic delivery of polypeptides through the eye

ABSTRACT

Compositions and methods for systemic delivery of polypeptides through the eyes are disclosed. The compositions include a systemically active polypeptide at a concentration such that the composition is substantially isotonic with tear fluid. The compositions may include a permeation-enhancing agent to aid systemic absorption of higher molecular weight polypeptides, as well as peptidase inhibitors. Therapeutically effective amounts of the polypeptide compositions can be administered to the eyes where the drug passes into the nasolacrimal duct and becomes absorbed into circulation.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a division of application Ser. No. 07/412,979 filedSep. 26, 1989, now U.S. Pat. No. 5,182,258, which is acontinuation-in-part application of Ser. No. 07/326,200, filed Nov. 20,1989, now abandoned.

TECHNICAL FIELD

The present invention relates generally to the administration oftherapeutic drugs and more particularly to the systemic delivery ofpolypeptides through the eyes.

BACKGROUND OF THE INVENTION

Therapeutic drugs have traditionally been administered orally or byinjection. However, several pharmaceuticals now being developed are noteasily administered via these methods. For example, many drugs,particularly peptides, are degraded by digestive enzymes and/or theacidity present in the gastrointestinal tract and cannot be takenorally. Additionally, many substances are not readily absorbed in thegastrointestinal tract due to the low permeability of the intestinalmembrane to hydrophilic compounds. Thus, these drugs must beadministered parenterally.

Injections, however, can be painful and must be given under sterileconditions to prevent the spread of AIDS and other infectious diseases.Furthermore, substances, such as insulin, administered subcataneously,show marked individual variability with respect to absorption See, e.g.,Galloway, J. A., et al., Diabetes Care (1981) 4:366-376. Additionally,repeated injections, often necessary to control such chronic diseases asdiabetes mellitus, can cause undesirable side effects such as scar-ring,irritation and localized edema. Therefore, patients often fail to complywith the strict regimen required to adequately treat such disorders,thus resulting in further medical complications. For instance, diabeticcataracts and retinopathy can occur in diabetics who fail to comply witha prescribed treatment plan.

Furthermore, several disorders are not amenable to self-help usinginjectables, although this is the most desirable method of treatment.For example, hypoglycemic crisis is preferably treated with intravenous,intramuscular or subcutaneous injections of glucagon or intravenousinjection of glucose solutions. Patients experiencing a hypoglycemicepisode cannot easily treat themselves with injections as their motorfunctions are impaired. However, treatment is crucial since prolongedhypoglycemia can lead to irreversible coma. Generally, patients mustresort to eating sugar candies, dextrose tablets or paste in order toraise the blood glucose concentration. This method is less thandesirable since the substances must travel to the intestine forabsorption and timing is crucial in such a crisis.

Drugs have also been delivered intranasally via nasal drops, spraysand/or inhalers. However, the amount of drug that reaches the nasalmucosa and ultimately becomes absorbed into the systemic circulation canbe less than optimal. Experimenters have used permeation-enhancingagents to aid absorption through the nasal mucosa. See, e.g., Hirai, S.,et al., Intl. J. Pharmaceutics (1981) 9:173-184; Monkhouse, D. C., andGroves, G. A., Aust. J. Pharm. (1967) 48:S70-S75; Moses, A. C.,Proceedings of Land O'Lake, (1986) 86, Merrimac, WI, Lecture Note, p. 6.

It is difficult, however, to achieve consistent drug distribution usingthese methods and delivery of a constant dose of drug intranasally isproblematic. Medical practitioners have attempted to use a metered-dosemechanical spray pump in an effort to achieve constant delivery.However, drugs so delivered have been found to be unevenly distributedin the septal wall with little being found in the lateral wall. Mygind,N., et al., Rhinology (1978) XVI: 79-88.

Other modes of administration include buccal, vaginal, rectal, dermaland tracheal delivery, none of which have been enthusiastically adopteddue to societal resistance and inconvenience.

The eye has several unique anatomical characteristics. It is protectedphysically by tough layers of blood-ocular barriers. Bito, L. Z., etal., The Ocular and Cerebrospinal Fluids, Academic Press, 1977, pp.229-243. The cornea is composed of an aqueous phase, the stroma,sandwiched by two lipid layers, the epithelium and endothelium,respectively. Most biological systems possess the opposite orientation.The nasolacrimal duct drains tears and other substances from the eye andis lined with absorptive mucosa. Thus, substances delivered into theocular cul-de-sac can enter the systemic circulation via thenasolacrimal system without significantly entering the eyes.

Drugs administered into the eye are generally intended for disorders ofthe eye itself and are not given to alleviate other systemicpathologies. However, much of the drug so administered is not absorbedby the eye, for reasons discussed above, and enters the systemiccirculation via the nasolacrimal system. Often, the concentrationsrequired to provide desired ophthalmic effects can result in undesirabledrug loads for the systemic circulation and sometimes toxicside-effects. For example, epinephrine has been used to treat eyedisorders however, the majority of instilled epinephrine enters thesystemic circulation as a result of absorption through the nasolacrimaldrainage system and has been reported to produce systemic alpha- andbeta-adrenergic side effects. McClure, D. A., General Pharmacology,Toxicology, and Clinical Experience, ACS Symposium Series, The AmericanChemical Society (1975), Number 14. Thus, the use of eye drops todeliver ophthalmic drugs has been problematic.

Attempts to deliver other drugs through the eye have been made but havegenerally been ineffective. For example, corneal absorption ofenkephalins in rabbits has been studied, and it was found that close to100% of the enkephalins recovered in the corneal epithelium were inhydrolyzed form due to peptidase cleavage thereof. Lee, V. H. L., etal., J. Ocular Pharmacol. (1986) 2:345.

Insulin was delivered to the rabbit conjunctiva with variable effects.Christie, C. D., and Hanzal, R. F., J. Clin. Invest. (1931) 10:787.Furthermore, the rabbit eye is proportinately larger than the human eyewhen compared to body size. Thus, enough drug can be administered to therabbit eye to elicit a systemic response. The human eye, on the otherhand, being very small in comparison to the body, generally cannotaccommodate the volume required in order to elicit an adequate response.Furthermore, when very concentrated drug solutions are administered tothe eye to avoid the use of large volumes, the ocular tonicity isdisrupted causing discomfort and eye irritation. For reviews of othersystems and mechanisms of ocular drug delivery see Lee, V. H. L.,Pharmaceutical Technology Apr. 1987:26 and Lee, V. H. L., PharmacyInternational (1985) 6:135.

Drugs can be administered to the eye using a variety of methods. Forexample, controlled-release formulations have been used to deliverophthalmic drugs to the eye for the treatment of eye diseases andinfections. Such formulations include matrix-type drug delivery systemssuch as hydrophilic soft contact lenses, soluble ocular inserts andscleral buckling materials. Capsule-type drug delivery systems, such asthe device Ocusert®, have been used for the delivery of theanti-glaucoma agent, pilocarpine, to the eye. Implantable siliconerubber devices have been used in the treatment of intraocularmalignancies. For a review of these sustained-release systems, see Ueno,N., et al., "Ocular Pharmacology of Drug Release Devices," in ControlledDrug Delivery, Stephen D. Bruck, ed., vol. II, chap. 4, CRC Press, Inc.(1983).

SUMMARY OF THE INVENTION

The present invention is based on the discovery that a wide range ofpolypeptides can be delivered efficiently via the eye to producesustained drug concentrations in the systemic circulation. This methodof administration is simple, convenient and painless. Furthermore, theblood concentrations of substances so administered are often sustainedfor longer periods of time when compared to intravenous administrationof a therapeutic dose of the same drug. Additionally, the dose of apolypeptide drug delivered via the eye can be readily controlled sincethe amount of formulation passing into the nasolacrimal duct isrelatively constant. The subject invention can also be used to deliverpolypeptide drugs directly to the eye to treat disorders therein.

In one embodiment, the present invention is directed to a pharmaceuticalcomposition for systemic delivery by ocular administration andabsorption in the nasolacrimal duct. The composition includes asystemically active polypeptide in a pharmaceutically acceptablevehicle, the polypeptide in the composition being present at aconcentration such that the composition is substantially isotonic withtear fluid. A permeation enhancing agent is also present to enhancenasolacrimal absorption of the polypeptide into the systemiccirculation.

Another embodiment of the present invention is directed to apharmaceutical composition for systemic delivery by ocularadministration and absorption in the nasolacrimal duct. The compositionincludes a systemically active polypeptide in an ocular delivery device.The ocular delivery device is formulated to release the polypeptide intotear fluid at a rate such that the concentration of polypeptide in thetear fluid does not significantly disrupt the tonicity of tear fluid.

In still another embodiment of the subject invention, a method isprovided for delivering a polypeptide drug systemically comprisingadministering a therapeutically effective concentration of the drugformulated in a pharmaceutically acceptable vehicle into the eye, theconcentration being less than the amount that will cause significantocular hypertonicity. An effective amount of a permeation-enhancingagent is coadministered with the drug, whereby the drug passes into thenasolacrimal duct where it is readily absorbed into systemiccirculation.

Another embodiment of the present invention provides a method fordelivering a polypeptide drug systemically comprising administering atherapeutically effective amount of the drug in an ocular deliverydevice The device is formulated to release the polypeptide drug intotear fluid at a rate such that the concentration of polypeptide in thetear fluid does not significantly disrupt the tonicity of the tear fluidThe released drug passes into the nasolacrimal duct where it is absorbedinto systemic circulation.

Further embodiments of the instant invention will readily occur to thoseof ordinary skill in the art.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1 depicts the absorption of insulin solutions into systemiccirculation via the eyes. Each point is a mean of 4 and 5 values for0.125% and 1.0% insulin concentrations, respectively. The bars representthe standard error of the mean ("SEM").

FIG. 2 shows a comparison of blood insulin concentration obtained viai.v. injection to that obtained by topical instillation through theeyes. Each point is a mean of 5 values and the bars represent SEM.

FIG. 3 demonstrates the absorption of various insulin concentrationswith the coadministration of 1% saponin into systemic circulationthrough the eyes. Each point is a mean of 6 values and bars representSEM.

FIG. 4 shows the effect of ocular administration of 1% insulin in 1%saponin on blood glucose concentrations in normal rabbits. Each point isa mean of 6 values and bars represent SEM.

FIG. 5 depicts the effect of ocular administration of 2% insulin in 1%saponin on blood glucose concentration in normal rabbits. Each point isa mean of 6 values and bars represent SEM.

FIG. 6 illustrates the effect of ocular administration of 5% insulin in1% saponin on blood glucose concentration in normal rabbits. Each pointis a mean of 6 values and bars represent SEM.

FIG. 7 demonstrates the effect of ocular administration of 1% insulin in1% saponin on blood glucose concentrations in alloxan-treated diabeticrabbits. Each point is a mean of 2 values.

FIG. 8 shows the effect of various concentrations of saponin onabsorption of a 1% insulin solution. Each point is a mean of 6 valuesand bars represent SEM.

FIG. 9 depicts the effect of 2% saponin on the uptake of a 1% insulinsolution and on blood glucose concentration. Each point is a mean of 6values and bars represent SEM.

FIG. 10 demonstrates the effect of 0.5% saponin on the uptake of a 1%insulin solution and on blood glucose concentration. Each point is amean of 6 values and bars represent SEM.

FIG. 11 illustrates the effect of 1% insulin on blood glucoseconcentration. Each point is a mean of 6 values and bars represent SEM.

FIG. 12 shows the effect of 1% saponin on the absorption of a 1% insulinsolution and blood glucose concentration. Each point is a mean of 6values and bars represent SEM.

FIG. 13 depicts the effect of an i.v. injection of 50 ug glucagon onblood glucose concentrations in normal rabbits. Bars represent SEM.

FIG. 14 demonstrates the effect of ocular administration of glucagon(0.2%) on blood glucose concentrations in normal rabbits. Bars representSEM.

FIG. 15 illustrates the effect of ocular administration of glucagon (1%)on blood glucose concentrations in normal rabbits. Bars represent SEM.

FIG. 16 shows the effect of ocular administration of glucagon (5%) onblood glucose concentrations in normal rabbits. Bars represent SEM.

FIG. 17 is a comparison of blood enkephalin-equivalent concentrationobtained via i.v. injection to that obtained by topical instillationthrough eyes. Bars represent SEM.

FIG. 18 shows the absorption of enkephalin equivalent into systemiccirculation after administration via the eyes. Bars represent SEM.

FIG. 19 is a comparison of blood TRH concentration obtained via i.v.injection to that obtained by topical instillation through the eyes.Bars represent SEM.

FIG. 20 depicts the absorption of TRH into systemic circulation throughthe eyes. Bars represent SEM.

FIG. 21 compares blood LHRH concentration obtained via i.v. injection tothat obtained by topical instillation through the eyes. Bars representSEM.

FIG. 22 shows the absorption of LHRH into systemic circulation throughthe eyes. Bars represent SEM.

FIG. 23 illustrates the enhancement of systemic absorption of LHRHthrough the eyes by the peptidase inhibitor Leu-Leu (5 mM).

FIG. 24 compares blood glucagon concentration after i.v. injection totopical instillation of glucagon. Bars represent SEM.

FIG. 25 depicts the absorption of glucagon into systemic circulationthrough the eyes. Bars represent SEM.

DETAILED DESCRIPTION

In describing the present invention, the following terms will beemployed, and are intended to be defined as indicated below.

A "systemically active polypeptide" is one that is delivered via thecirculation to act at a site remote from its entry into the body.Additionally, such a polypeptide should retain its biological activityfor an effective period of time when present in the circulatory systemof the subject to which it is administered. The polypeptide can berecombinantly-derived, purified, or present in compositions of thesubject invention in crude form.

The terms "polypeptide" and/or "polypeptide drug" are used in theirbroadest sense, i.e., any polymer of amino acids (dipeptide or greater)linked through peptide bonds. Thus, the terms include proteins,oligopeptides, protein fragments, analogs, muteins, fusion proteins andthe like. These terms also encompass amino acid polymers as describedabove that include additional moieties. Thus, the terms "polypeptide"and "polypeptide drug" include glycoproteins, lipoproteins,phosphoproteins, metalloproteins, nucleoproteins, as well as otherconjugated proteins.

A "permeation-enhancing agent" is an agent that increases the amount ofnasolacrimal absorption into systemic circulation of a substancecoadministered therewith. Representative enhancers include surfactants,including cationic, anionic and nonionic detergents, bile salts andacids, chelating agents, fusidic acid derivatives, among others. Suchagents are discussed more fully below.

A "peptidase inhibitor" is a substance that substantially inhibits theenzymatic activity of peptidases.

A composition of the present invention is "substantially isotonic withtear fluid" when it has substantially the same osmotic pressure as thatfound in the tear fluid. Generally, a solution with a tonicity equal tothat of a 0.9% sodium chloride solution is isotonic. However, solutionsequivalent in tonicity to a range of 0.5% to 1.8% sodium chloride willalso be tolerated by the eye and will find use with the presentinvention.

By "hypertonicity" is meant that state where the osmolality of thesubject fluid is increased above that found in the isotonic state."Significant" hypertonicity refers to a hypertonic state that woulddamage the anatomical constituents of the eye and/or surrounding tissue.Generally, solutions with a tonicity greater than a 1.8% sodium chloridesolution would be considered "significantly hypertonic."

The term "therapeutically effective amount" as used herein refers to theamount of polypeptide drug sufficient to elicit at least a desiredthreshold response to the drug in a subject to which the drug isadministered. The precise amount will vary with the particularpolypeptide drug employed, the age and condition of the subject treated,and the nature and severity of the condition. However, therapeuticallyeffective amounts of many drugs are known or can be readily determinedby one of skill in the art.

An "effective amount" of a permeation-enhancing agent is an amount thatwill increase nasolacrimal absorption of a coadministered drug so thatblood levels of the drug can reach a concentration required to elicit athreshold response thereto without significantly disrupting theisotonicity of the tear fluid.

The blood concentration of a polypeptide drug administered according tothe present invention is dependent on (1) the concentration of drugdelivered and (2) the amount of drug absorbed via the conjunctiva andnasolacrimal system. Higher molecular weight polypeptides are not asreadily absorbed as smaller polypeptides. Thus, a larger amount of thehigher molecular weight polypeptide must be administered in order toachieve adequate blood concentrations. However, the delivery of apolypeptide in an amount greater than about 10% (w/v) generally resultsin a significantly hypertonic solution, not suitable for insertion intothe eye. A permeation enhancer as described above can be used toincrease absorption of higher molecular weight polypeptides so that theamount of drug delivered will not significantly disturb the isotonicenvironment of the eye. The amount of enhancer used should not be somuch as to cause eye irritation. An effective amount of enhancer willvary depending on the enhancing agent used, however, concentrations onthe order of 0.1%-2% (w/v) will find use with the present invention.

A "pharmaceutical composition", according to the present invention, isone that includes a polypeptide drug. The composition may also contain apharmaceutically acceptable vehicle. The vehicle should be compatiblewith the active ingredient of the pharmaceutical composition. Suitablevehicles for ocular use are, for example, sterile isotonic solutionssuch as isotonic sodium chloride or boric acid solutions. These vehiclestypically contain sodium chloride or boric acid, respectively, as wellas benzalkonium chloride and sterile distilled or purified water. Alsouseful is phosphate buffered saline (PBS), pH 7.4. Other suitablevehicular constituents include phenylmercuric nitrate, sodium sulfate,sodium sulfite, sodium phosphate and monosodium phosphate.

The compositions may also contain auxiliary substances i.e.antimicrobial agents such as chlorobutanol, parabans or organicmercurial compounds; pH adjusting agents such as sodium hydroxide,hydrochloric acid or sulfuric acid; and viscosity increasing agents suchas methylcellulose. One of ordinary skill in the art will easily findsubstitutions for the above auxiliary substances. The final compositionshould be sterile, essentially free of foreign particles, and have a pHthat allows for optimum drug stability. Generally pH values in the rangeof 5-8 will find use with the subject compositions. Preferably, the pHwill be as close to the pH of tear fluid, i.e., 7.4, as possible.

Typically, the compositions of the subject invention are prepared assolutions, suspensions, ointments, gels, or ocular delivery devices suchas drug-impregnated solid carriers that are inserted into the eye. Ifsuch a carrier is used, the above-mentioned vehicles are unnecessary. Avariety of polymers can be used to formulate ophthalmic drug carriers.Saettone, M. F., et al., J. Pharm. Pharmocol. (1984) 36:229, and Park,K., et al., in Recent Advances in Drug Delivery Systems, James M.Anderson and sung Wan Kiy, eds, Plenum press (1984), pp. 163-183,describe such polymers, the disclosures of which are incorporated hereinby reference in their entirety. Drug release is generally effected viadissolution or bioerosion of the polymer, osmosis, or combinationsthereof. The device should be formulated to release the polypeptide at arate that does not significantly disrupt the tonicity of tear fluid.

More specifically, several matrix-type delivery systems can be used withthe subject invention. These systems are described in detail in Ueno,N., et al., supra, the disclosure of which is incorporated herein byreference in its entirety. Such systems include hydrophilic soft contactlenses impregnated or soaked with the desired drug, as well asbiodegradable or soluble devices that need not be removed afterplacement in the eye. These soluble ocular inserts can be composed ofany degradable substance that can be tolerated by the eye and that iscompatible with the drug to be administered. Such substances include butare not limited to poly(vinyl alcohol), polymers and copolymers ofpolyacrylamide, ethylacrylate and vinylpyrrolidone, as well ascrosslinked polypeptides or polysaccharides, such as chitin.

Capsule-type delivery systems will also find use with the instantinvention. These systems, described in Ueno, N., et al., supra, utilizepolymer membranes to control the release of the drug in question. Thesedevices are particularly useful for the delivery of hydrophilic drugs.Hydrophobic drugs can be administered via a silicone rubber device suchas described in Ueno, N., et al., supra.

Ophthalmic ointments will include a base, generally composed of whitepetrolatum and mineral oil, often with anhydrous lanolin.Polyethylene-mineral oil gel is also satisfactory, as are othersubstances that are nonirritating to the eye, permit diffusion of thedrug into the ocular fluid, and retain activity of the medicament for areasonable period of time under storage conditions. If suspensions areused, the particle sizes therein should be less than 10 um to minimizeeye irritation. Furthermore, if solutions or suspensions are used, theamount delivered to the patient should not exceed 75 ul, preferably 50ul or less, to avoid excessive spillage from the eye.

A wide range of polypeptides will find use in the subject compositions.Exemplary polypeptides and their approximate molecular weights can beseen in Table 1. The invention is particularly useful for theadministration of such peptide hormones as insulin, thyrotrophinreleasing hormone (TRH), luteinizing hormone releasing hormone (LHRH),oxytocin, vasopressin, lypressin, growth hormone releasing factor,gonadotropin releasing hormone, somatotropin, somatostatin, secretin,calcitonin, among others; peptide analgesics such as but not limited toenkephalins including leu- and met- enkephalin and endorphins such asalpha-neoendorphin, beta-neoendorphin, dynorphin A and dynorphin B;glucagon, concanavalin, ribonuclease, lysozyme and ACTH. Generally, anypeptide capable of sustaining biological activity when present in thesystemic circulation will find use with the subject invention.

The amount of peptide present in the compositions will vary according tothe peptide used, condition, and age and size of the subject beingtreated. Doses can be readily determined by one of skill in the art.Generally, however, the concentration of peptide administered should notexceed 10% (w/v) of the composition, less the isotonic environment ofthe eye will be significantly disturbed. Thus, since the delivery of 50ul or less of the peptide composition is preferred, the amount ofpeptide present int he composition will generally be 5 mg or less (50 ulof a 10% solution).

A permeation enhancer can be coadministered with the peptide drug,either simultaneously as part of the composition, or shortly before orafter delivery of the peptide. The use of a permeation enhancer aidsabsorption of the drug into systemic circulation via the mucous membraneand allows the delivery of less concentrated substances to the eye.Thus, a wider range of peptide drugs can be administered withoutdisturbing the isotonicity of the tear fluid. Such enhancers areparticularly useful with

                  TABLE 1                                                         ______________________________________                                        Exemplary Polypeptides for use with                                           the Subject Invention                                                                                    Molecular                                          Polypeptide                Weight                                             ______________________________________                                        Thyrotrophin Releasing Hormone (TRH)                                                                       360                                              Leucine-Enkephalin           600                                              Methionine-Enkephalin        600                                              Somatotropin                 800                                              Oxytocin                   1,000                                              Vasopressin                1,000                                              Lypressin                  1,000                                              alpha-Neoendorphin         1,100                                              beta-Neoendorphin          1,100                                              Luteinizing Hormone Releasing Hormone (LHRH)                                                             1,200                                              Dynorphin A                1,400                                              Dynorphin B                1,400                                              Somatostatin               1,650                                              Secretin                   3,000                                              Calcitonin                 3,400                                              Glucagon                   3,500                                              ACTH                       4,500                                              Growth Hormone Releasing Hormone                                                                         4,800                                              Insulin                    6,000                                              Concanavalin               12,000                                             Ribonuclease               13,000                                             Lysozyme                   15,000                                             ______________________________________                                    

polypeptides having molecular weights exceeding 5000.

Suitable enhancing agents include either alone or in combination,surfactants such as saponins, polyoxyethylene, polyoxyethylene ethers offatty acids such as polyoxyethylene 4-, 9-, 10-, and 23-lauryl ether,polyoxyethylene 10- and 20-cetyl ether, polyoxyethylene 10- and20-stearyl ether, sorbitan monooleate, sorbitan monolaurate,polyoxyethylene monolaurate, polyoxyethylene sorbitans such aspolyoxyethylene sorbitan monolaurate, decamethonium, decamethoniumbromide, and dodecyltrimethylammonium bromide; chelators such as EDTAand disodium EDTA; bile salts and acids such as cholic acid, deoxycholicacid, glycocholic acid, glycodeoxycholic acid, taurocholic acid,taurodeoxycholic acid, sodium cholate, sodium glycocholate,glycocholate, sodium deoxycholate, sodium taurocholate, sodiumglycodeoxycholate, sodium taurodeoxycholate, chenodeoxycholic acid, andurosdeoxycholic acid; fusidic acid derivatives, glycyrrhizic acid, andammonium glycyrrhizide, with saponin EDTA, fusidic acid, polyoxyethylene9-lauryl ether, polyoxyethylene 20-stearylether, and glycocholate beingpreferred.

The concentration of enhancer administered should be the minimum amountneeded to sufficiently increase absorption through the mucous membrancesof the nasolacrimal duct so as to avoid use of high concentrations ofdrug that might cause irritation to the eye. Generally, concentrationsranging from 0.1% to 5% (w/v), more preferably 00.25% to 2%, will finduse with the subject compositions.

Peptidase inhibitors may also be coadministered, either as constituentsof the subject compositions, or shortly before or after delivery of thepeptide drug, to inhibit the activity of peptidases present in the eye.Representative peptidase inhibitors include Leu-Leu, bestain,D,L-thiorphan, puromycin, captopril, bacitracin, phenylmethyl sulfonylfluoride, leupectin, pepstatin A, and aprotinin. The concentration ofinhibitor present will depend on the particular peptidase inhibitorused. Concentrations for representative inhibitors are given below inthe examples.

Generally, the compositions of the present invention will beadministered as often as needed to sustain the proper bloodconcentration of the drug being so delivered. Systemic bloodconcentrations can be monitored and the amount and frequency ofadministration determined accordingly by one of ordinary skill in theart. The compositions can be formulated to allow for the slow,controlled release of the peptide drug into the eye.

Below are examples of specific embodiments for carrying out the presentinvention. The examples are offered for illustrative purposes only, andare not intended to limit the scope of the present invention in any way.

EXAMPLES Example 1 Systemic Delivery of Insulin through the Eyes A.Materials

Commercially available U-500 regular porcine insulin (Iletin® II,containing 500 U/ml) was obtained from Eli Lilly (Indianapolis, IN).Radioactive ¹²⁵ I-insulin (porcine) was purchased from DuPont NENResearch Products (Wilmington, DE, with a specific activity of 94.9uCi/ug). Alloxan monohydrate, saponin, insulin and leucine-leucine werepurchased from Sigma Chemical Company (St. Louis, MO). all peptides weredissolved in phosphate buffered saline (PBS) at a pH of 7.4 to a finalconcentration of 0.125%, 1%, 2% or 5% (w/v) along with the radioactivepeptides (0.625 uCi/25 ul). A radioactive solution of 1%, 2% and 5%insulin in 1% saponin was also prepared in PBS.

B. Methods

To induce hyperglycemia in rabbits, a 10% solution of alloxanmonohydrate was prepared in citrate-phosphate buffer at a pH of 3.5-4.0.The solution was sterilized by filtering through a Millipore filter andwas kept in the refrigerator before use. New Zealand white rabbitsweighing 2-3 kg were fasted for 24 hrs before injection with alloxan.The animals were anesthetized with ketamine HCl and zylazine during theinjection of 10% alloxan (100 mg/kg) into the marginal ear vein at arate of 0.5 ml/min. Hyperglycemia, with blood glucose over 300 mg %,developed 3 days after alloxan injection. Blood glucose levels weredetermined before, and at least twice weekly after alloxan injection,using one drop of fresh whole blood collected by venipuncture andapplied to a GLUCOSCAN Test Strip and read one minute later by aGLUCOSCAN™ 2000 Meter (Lifescan Inc.; Mountain View, CA). Those animalswere selected in which the blood glucose levels were greater than 300 mg% for at least two weeks. The control animals (normal) had blood glucoselevels of 100 to 150 mg %.

For insulin absorption experiments, normal New Zealand white femalerabbits weighing 2.0-3.0 kg were used. The rabbits were anesthetizedwith 30 mg/kg pentobarbital sodium intravenously through the marginalear vein. Additional pentobarbital sodium (1 mg/kg/hr) was giventhroughout the experiment to maintain anesthesia. The femoral artery wascannulated with polyethylene tubing (PE-90) for the collection of bloodsamples and for the replacement of blood volume with heparinized saline.Twenty-five microliters of insulin solution was instilled into the lefteye of the rabbit. 1 ml aliquot of blood was collected from the femoralartery at 0, 5, 10, 20, 30, 60, 90, 120, 150, 180 min and every 1 hrthereafter for a total time period of 12 hrs. The radioactivity of bloodsamples was counted in a gamma counter (Packard Auto Gamma 500, Packardinstrument Company, Downers Grove, IL), and the concentration of insulinabsorbed was calculated. At the end of blood collection, the rabbitswere euthanized with sodium pentobarbital. The left eye was enucleatedand dissected. The cornea, iris, ciliary body, lens, retina and choroidwere isolated, and the wet weight of tissues promptly measured. Theradioactivity of each tissue sample was determined with a PackardAuto-Gamma Counter and the concentration of insulin in each tissuecalculated. The internal standard was prepared by using the dilutedinsulin (0.0025 uCi/10 ul).

The i.v. injection of insulin at 50 ug (with 0.125 uCi/50 ul ofradioactivity) was also carried out. One milliliter of blood sample wascollected at 0, 2, 4, 6, 10, 20, 30, 60 90, 120, 150, 180, 240, and 300minute intervals. The blood volume was replaced by an equal volume ofheparinized normal saline. The radioactivity of blood samples wasdetermined with a gamma counter. The concentration of insulin in theblood was expressed in ng/ml. For insulin action in diabetic animals,alloxantreated rabbits were used. Rabbits were anesthetized with 30mg/kg pentobarbital sodium intravenously and were then maintainedanesthetized with an additional dose of pentobarbital sodium at a rateof 1 mg/kg/hr. Radioactive insulin solution (1% insulin in 1% saponin)was instilled (25 ul for normal rabbits and 50 ul for diabetic animalswhich were equivalent to approximately 6 U and 12 U insulin,respectively) into the left eyes of 12 hr fasted animals, and 1 mlaliquot of blood samples was collected from the cannulated femoralartery at time intervals specified in the RESULTS section. The bloodinsulin level was determined in a gamma counter (Packard Auto-Gamma 500,Packard Instrument Company, Downers Grove, IL). The blood glucose wasmeasured on a GLUCOSCAN™ 2000 Meter.

C. Results

All data were analyzed with Student's t-test for two values and analysisof variance for more than two values. Each value was expressed asmean±standard error of the mean. A p value of 0.05 or less wasconsidered significant.

When 0.125% insulin solution was instilled into eyes, it reached 1.3ng/ml in 11 hrs (FIG. 1). A 1% insulin solution took 8 hrs to reach aplateau concentration of 9 ng/ml (FIG. 1). A 5% insulin solution tookonly 2.5 hrs to reach a plateau at 24 ng/ml (FIG. 2). These resultsindicate a good dose-absorption relationship of insulin in the range of0.125%-5% solutions (FIGS. 1 and 2).

When 50 ug of insulin was injected intravenously, the bloodconcentration of insulin could be maintained at 27 ng/ml (FIG. 2) whichwas about the same as that caused by an eye drop of 5% (25 ul) whichmaintained blood concentration at 25 ng/ml (FIG. 2). These resultsindicate that ocular administration is comparable with i.v. injection ofinsulin.

To test whether the permeation enhancer saponin increased insulinabsorption into systemic circulation, 25 ul of 1%, 2% and 5% insulinplus 1% saponin solution were instilled into eyes as above. Bloodinsulin concentrations reached 63 ng/ml, 89 ng/ml and 195 ng/ml,respectively, in the systemic circulation (FIG. 3). These resultsindicate that 1% saponin can enhance insulin absorption by at least7-fold (FIGS. 1, 2 and 3).

A change of pH from 5 to 8 did not affect insulin absorption.Furthermore, the addition of the peptidase inhibitor leucine-leucine didnot significantly improve insulin absorption. Without being bound by anyparticular theory, this result is probably due to the fact thatpeptidase activity in conjunctiva and tears is low as opposed topeptidase activity in cornal epithelium and iris-ciliary body. Thus,peptides that are not absorbed by the eye but rather remain in thecul-de-sac for absorption into systemic circulation appear relativelysafe from degradation.

Most importantly, 25 ul of 1% insulin in 1% saponin was able to lowerthe blood glucose of 12 hr fasted normal animals from 102 mg % to 50 mg% in an hour. Blood glucose concentrations remained low for at least 3hrs (FIG. 4). Higher concentrations of insulin (2% and 5%) in 1% saponinwere able to depress the blood glucose concentrations to even lowerlevels (approximately 40-45 mg %) for longer periods of time (FIGS. 5and 6). When 50 ul of 1% insulin in 1% saponin was instilled into theeyes of alloxan-treated diabetic animals, the blood glucose of 12 hrfasted animals was reduced from 370 mg % to 185 mg % in 90 min andfurther to 75 mg % in another 150 min (FIG. 7). Table 2 illustrates theamount of insulin remaining in various eye tissues at the end of a 12 hrexperiment. As can be seen, the amount remaining ranged from0.028-0.049% of the total amount of insulin instilled into the eyes.

                                      TABLE 2                                     __________________________________________________________________________    The amount of insulin remaining in eye tissues at the end of 12 hr            experiment                                                                           Residual amount of insulin remaining in tissues (ng/tissues)                  0.125% insulin (n = 4)                                                                       1% insulin (n = 5)                                                                           5% insulin (n = 5)                       Tissue Amount % of instilled                                                                        Amount % of instilled                                                                        Amount  % of instilled                   __________________________________________________________________________    Cornea  5.34 ± 1.56                                                                      0.0171 ± 0.0050                                                                    24.68 ± 4.72                                                                      0.0099 ± 0.0019                                                                    130.94 ± 24.04                                                                     0.0105 ± 0.0019               Iris    0.74 ± 0.27                                                                      0.0024 ± 0.0009                                                                     3.59 ± 1.11                                                                      0.0014 ± 0.0004                                                                     18.88 ± 3.23                                                                      0.0015 ± 0.0003               Ciliary-Body                                                                          0.82 ± 0.25                                                                      0.0026 ± 0.0008                                                                     3.98 ± 2.94                                                                      0.0016 ± 0.0012                                                                     24.13 ± 4.45                                                                      0.0019 ± 0.0004               Lens    2.95 ± 0.95                                                                      0.0094 ± 0.0030                                                                    11.12 ± 4.84                                                                      0.0045 ± 0.0019                                                                     36.80 ± 5.66                                                                      0.0029 ± 0.0005               Retina  2.18 ± 0.63                                                                      0.0070 ±  0.0020                                                                    8.52 ± 4.54                                                                      0.0034 ± 0.0018                                                                     48.75 ± 9.99                                                                      0.0039 ± 0.0008               Choroid                                                                               3.40 ± 1.11                                                                      0.0109 ± 0.0036                                                                    19.09 ± 15.05                                                                     0.0076 ± 0.0060                                                                     94.39 ± 26.03                                                                     0.0076 ± 0.0021               TOTAL  15.43 ng                                                                             0.0494% 70.98 ng                                                                             0.0284% 353.89 ng                                                                             0.0283%                          __________________________________________________________________________

This study demonstrates that the ocular nasolacrimal system is afeasible route for insulin absorption into the systemic circulationresulting in a concomitant reduction of blood glucose concentrations inalloxantreated diabetic, as well as normal, animals. Systemic insulinabsorption through the eyes can be improved by an absorption enhancer,saponin, to facilitate its absorption. With 1% saponin, the bloodconcentration of insulin can reach at least 7-fold higher levels thanwithout saponin; the blood glucose concentrations can also reach lowerlevels for longer periods.

EXAMPLE 2 Increased Systemic Insulin Absorption Using PermeationEnhancers

To test the ability of various permeation enhancers to increase insulinabsorption, the following experiment was conducted.

A. Materials

Insulin, sodium glycocholate, decamethonium bromide,polyoxyethylene-9-lauryl ether (BL-9), polyoxyethylene sorbitanmonolaurate (tween 20), saponin, EDTA, and sodium fusidate werepurchased from Sigma Chemical Company (St. Louis, MO). ¹²⁵ I-insulin(Spec. Act. 94 uCi/ug) was purchased from DuPont NEN Research Products(Wilmington, DE). All agents were dissolved in phosphate buffered saline(PBS) at pH 7.4 along with radioactive insulin.

B. Methods

New Zealand white female rabbits weighing 2.0-3.0 kg were used. Therabbits were anesthetized with 30 mg/kg were used. The rabbits wereanesthetized with 30 mg/kg pentobarbital sodium intravenously throughthe marginal ear vein. Additional pentobarbital sodium (1 mg/kg/hr) wasgiven throughout the experiment to maintain anesthesia. The femoralartery was cannulated with polyethylene tubing (PE-90) for thecollection of blood samples and for replacement of blood volume withheparinized (50 U/ml) normal saline. 25 ul of radioactive insulinsolution (0.625 uCi/25 ul) was instilled into the left eye of therabbit. 1 ml aliquot of blood was collected from the femoral artery at0, 5, 10, 20, 30, 60, 90, 120, 150 and 180 min and every 1 hr thereafterfor a total time period of 6 hrs. The radioactivity of blood samples wascounted in a gamma counter (Packard Auto-Gamma 500, Packard InstrumentCompany, Downers Grove, IL) and the concentration of insulin absorbedwas calculated. Blood glucose levels were determined using one drop offresh whole blood collected by venipuncture and applied to a GLUCOSCANTest Strip and read one minute later by a GLUCOSCAN™ 2000 Meter(Lifescan inc., Mountain view, CA). At the end of blood collection, therabbits were euthanized with an overdose of sodium pentobarbital. Theinternal standard was prepared by using the diluted correspondinginsulin (0.0025 uCi/10 ul). The concentration of insulin equivalent inthe blood was expressed in ng/ml.

C. Results

All data were analyzed with Student's t-test for two values and analysisof variance for more than two values. Each value was expressed asmean±standard error of the mean. A p value of 0.05 or less wasconsidered significant.

When 1% insulin was instilled to the eye, the blood concentrationreached only 6.5 ng/ml (FIG. 10).

Glycocholate is an anionic detergent and a bile salt. 1% enhanced theinsulin absorption approximately 2-fold and reduced the blood glucosefrom 108 mg % to 94 mg % (Table 3).

Decamethonium is a cationic detergent which did not significantlyimprove the insulin absorption nor reduce the blood glucose (Table 3).

Polyoxyethylene 9-lauryl ether (BL-9) is a nonionic detergent 1% ofwhich enhanced insulin penetration at least 3-fold and markedly reducedthe blood glucose from 97 mg % to 56 mg % (Table 3).

Tween 20 (1%) behaved similarly with decamethonium, showing only aslight increase in insulin absorption and a minute reduction in bloodglucose concentration (Table 3).

EDTA is a chelating agent which can loosen intercellular tight junction.1% of EDTA increased insulin penetration 3-fold and reduced bloodglucose concentration significantly from 94 mg % to 64 mg % (Table 3).

Fusidic acid (1%) showed marked enhancement of insulin absorption(approximately 4-fold) and significant reduction in blood glucoseconcentration from 90 mg % to 51 mg % (Table 3).

among all agents tested saponin (1%) showed the most potent efficacy toenhance insulin absorption (approximately 10-fold) and reduce bloodglucose form 102 mg % to 41 mg % (FIG. 12; Table 3).

                  TABLE 3                                                         ______________________________________                                        Effects of Absorption Enhancers on Insulin                                    Penetration and Blood Glucose Concentrations                                                       Blood Glucose                                                                              % Change                                    Absorption                                                                             Insulin peak                                                                              Concentration                                                                              in Blood                                    Enhancer Concentration                                                                             (mg %)       Glucose Con-                                (1% Solution)                                                                          (ng/ml)     Before   After centration                                ______________________________________                                        Control   6.5 ± 0.8                                                                             93 ± 2                                                                              94 ± 3                                                                           no change                                 Saponin  63.0 ± 6.3                                                                             102 ± 4                                                                             41 ± 2                                                                           60                                        Fusidic acid                                                                           26.5 ± 4.5                                                                             90 ± 3                                                                              51 ± 2                                                                           43                                        BL-9     20.0 ± 1.8                                                                             97 ± 2                                                                              56 ± 3                                                                           42                                        EDTA     22.5 ± 5.7                                                                             94 ± 3                                                                              64 ± 5                                                                           32                                        Glycocholate                                                                           13.3 ± 1.4                                                                             108 ± 7                                                                             94 ± 4                                                                           13                                        Deca-    10.8 ± 1.8                                                                             90 ± 3                                                                              83 ± 2                                                                            8                                        methonium                                                                     Tween 20  8.8 ± 0.9                                                                             90 ± 3                                                                              77 ± 9                                                                           14                                        ______________________________________                                         The eye drops contained 1% insulin plus 1% absorption enhancer.          

Various concentrations of saponin were tested to study the dose-potencyeffect of saponin on blood glucose levels and enhanced insulinabsorption. It was found that an increase of saponin concentrationbeyond 1% did not significantly increase insulin absorption nor reduceblood glucose concentration (FIG. 8).

However, reduction of saponin concentration from 1% to 0.5% did reducethe ability to enhance insulin absorption and to decrease blood glucoseconcentration (FIG. 8). The blood glucose concentrations were lowered toabout the same extent with 1% insulin plus either 1% or 2% saponin, from95 mg% to 40 mg% (FIGS. 9 and 12). When 1% insulin alone was instilled,the insulin concentration in the blood (6.5 ng/ml) did not reach theeffective concentration (at least 20 ng/ml) to lower the blood glucoseconcentration (FIG. 11). Without absorption enhancers, an insulinconcentration of at least 5% would be required to lower the bloodglucose concentration.

Absorption enhancers not only increase insulin uptake into systemiccirculation but also into eyeballs as can be seen from Table 3. Therewas a good correlation between uptake of insulin into systemiccirculation and the eyeball (Tables 3 and 4).

Thus, permeation absorption enhancers are useful in increasing systemicabsorption. Although the above studies were performed using insulinonly, these enhancers also find utility with other peptide drugs(described below), particularly larger peptides.

                  TABLE 4                                                         ______________________________________                                        Effects of Absorption Enhancers on the Penetration                            of Insulin into Eyeballs                                                      1% insulin  % of insulin taken                                                                         Absolute amount of                                   plus enhancer                                                                             up by the eyeball                                                                          insulin in the eyeball                               ______________________________________                                        1% Saponin  1.9186       4796 ng                                              0.5% Saponin                                                                              0.6733       1683 ng                                              1% Fusidic A                                                                              0.4767       1191 ng                                              1% BL-9     0.5221       1305 ng                                              1% EDTA     0.1684       420 ng                                               1% Glycocholate                                                                           0.0804       200 ng                                               1% C10      0.0683       170 ng                                               1% Tween 20 0.0771       192 ng                                               Control     0.0744       185 ng                                               (1% insulin only)                                                             ______________________________________                                    

EXAMPLE 3 Systemic Delivery of Glucagon Through the Eyes

Glucagon is useful in the treatment of insulin-induced hypoglycemia. Totest whether glucagon could be effectively administered through theeyes, the following experiment was conducted as described in Chiou, C.Y. and Chuang, C. Y. J. Ocular Pharm. (1988) 4:179-186.

A. Materials

Glucagon was purchased from Sigma Chemical Company (St. Louis, MO).Radioactive ¹²⁵ I-glucagon was obtained from DuPont NEN ResearchProducts (Wilmington, DE, with a specific activity of 131 mCi/mg).

Glucagon was dissolved in phosphate buffered saline (PBS) at pH 7.4 to afinal concentration of 0.2% 1% or 5% (w/v) along with the radioactiveglucagon (0.625 uCi/25 ul).

B. Methods

New Zealand white female rabbits weighing 2.0-3.0 kg were treated as inExample 2B above, with the exception that 25 ul of radioactive glucagonsolution (0.625 uCi/25 ul) was used. At the end of blood collection, therabbits were euthanized with an overdose of sodium pentobarbital. Theleft eye was enucleated and dissected. The cornea, iris, ciliary body,lens, retina and choroid were isolated, and the wet weight of tissuespromptly weighed. The radioactivity of each tissue sample was alsodetermined with a Packard Auto-Gamma Counter and the concentration ofglucagon in each tissue calculated. The internal standard was preparedby using the diluted corresponding glucagon (0.0025 uCi/10 ul). An i.v.injection of glucagon at 50 ug (with 0.125 uCi/50 ul of radioactivity)was also carried out. One milliliter of blood sample was collected at 0,15, 30, 60, 90, 120, 150, 180, 240, 300, and 360 minutes intervals. Theblood volume was replaced by an equal volume of heparinized normalsaline. The radio-activity of the blood sample was determined with agamma counter. The blood glucose was measured on a GLUCOSCAN™2000 Meter.The concentration of glucagon in the blood was expressed in ng/ml.

C. Results

All data were analyzed with the Student's t-test for two values andvariance analyzed for more than two values. Each value was expressed asmean±standard error of the mean. A p value of 0.05 or less wasconsidered significant.

The therapeutic dose of glucagon is 1 mg/70 kg or 14.29 ug/kg.Therefore, 50 ug of glucagon was injected i.v. into rabbits ofapproximately 3 kg (FIG. 13). The blood concentration of glucagondeclined rapidly in 30 min and then decreased to 12.5 ng/ml in 6 hrs.The blood glucose concentration increased rapidly after the i.v.injection of glucagon, to reach 235 mg % from 125 mg % as expected. Theblood glucose peaked at 30 min after i.v. injection and then reachedbottom at 120 min after the injection (FIG. 13).

When 25 ul of 0.2% glucagon (equivalent to 50 ug) was instilled into theleft eye, it raised blood glucose effectively to 204 mg % from 104 mg %in 30 min (FIG. 21). The blood glucose concentration came down to 116 mg% in 2 hrs and remained at the same level for the rest of theexperiments (6 hrs). The blood glucagon concentration increasedgradually and reached the plateau at 8 ng/ ml in 2 hrs and remainedthere for the rest of the experiments (FIG. 14).

When 25 ul of 1% glucagon (equivalent to 250 ug) was instilled into theleft eye, it raised blood glucose concentration to 248 mg % from 103 mg% in 30 min and then remained high for another 60 min (FIG. 15). Theblood glucose concentration of glucagon reached the plateau at 37 ng/mlin 2 hrs and then remained high during the rest of the experiments (FIG.15).

When 25 ul of 5% glucagon (equivalent to 1250 ug) was instilled, theblood concentration of glucagon kept rising during the experimentalperiod of 6 hrs to reach 130 ng/ml (FIG. 16). The blood glucoseconcentration rose quickly to 276 mg % from 130 mg % in 60 min andremained high for the next 90 min. The blood glucose concentrationdeclined gradually thereafter to reach 160 mg % at the end of the 6 hrexperiment period (FIG. 16).

The amount of glucagon taken up into the eyes was minimal at the end ofthe 6 hr experimental period. Most of the glucagon detected remained inthe cornea where the eye drops contacted directly (Table 5). The totalamount of glucagon remaining in the eye tissues except cornea was only0.105%, 0.103% and 0.123% of the absolute quantity of 0.2%, 1% and 5%solutions instilled into the eyes, respectively. Among cornea,iris-ciliary body, lens, retina and choroid, the great majority 71%, 84%and 87%, respectively, remained in the cornea.

                                      TABLE 5                                     __________________________________________________________________________    The amount of glucagon remaining in eye tissues at the end of 6 hr            experiment                                                                           Residual amount of glucagon remaining in tissues (ng/tissues)                 0.2% (n = 7     1% (n = 5)       5% (n = 5)                            Tissue Amount  % of instilled                                                                        Amount   % of instilled                                                                        Amount    % of instilled              __________________________________________________________________________    Cornea 128.26 ± 42.99                                                                     0.2565 ± 0.0860                                                                    1314.97 ± 364.89                                                                    0.5260 ± 0.1460                                                                     9911.85 ± 2625.87                                                                   0.7930 ± 0.2101          Iris    10.12 ± 3.99                                                                      0.0202 ± 0.0080                                                                     77.22 ± 25.77                                                                      0.0309 ± 0.0103                                                                     402.44 ± 90.51                                                                      0.0322 ± 0.0073          Ciliary-Body                                                                          3.68 ± 1.23                                                                       0.0074 ± 0.0025                                                                     25.51 ± 7.63                                                                       0.0102 ± 0.0031                                                                     153.14 ± 38.42                                                                      0.0123 ± 0.0031          Lens    35.05 ± 15.09                                                                     0.0701 ± 0.0302                                                                     135.54 ± 35.75                                                                     0.0542 ± 0.0143                                                                     871.05 ± 210.82                                                                     0.0697 ± 0.0169          Retina  1.42 ± 0.34                                                                       0.0028 ± 0.0007                                                                       6.15 ± 1.90                                                                      0.0025 ± 0.0008                                                                      56.28 ± 23.15                                                                      0.0045 ± 0.0019          Choroid                                                                               2.11 ± 0.54                                                                       0.0042 ± 0.0011                                                                     13.07 ± 4.60                                                                       0.0052 ± 0.0018                                                                      57.79 ± 13.07                                                                      0.0046 ± 0.0011          Total  180.64 ng                                                                             0.3612% 1572.46 ng                                                                             0.6290% 11452.55 ng                                                                             0.9163%                     __________________________________________________________________________

EXAMPLE 4 Systemic Delivery of Enkephalin Peptide Through Eyes

Leu-enkephalin is a peptide demonstrating analgesic action and isreadily available commercially. To test whether enkephalin peptide couldbe effectively administered via the eyes, the method described in Chiou,G.C.Y. et al., Life Sciences (1988) 43:509-514 was followed.

A. Materials

Leucine-enkephalin (5-L-leucine), [¹²⁵ I]-(specific activity of 2200Ci/mmole) was obtained commercially from DuPont NEN Research Products(Wilmington, DE). Enkephalin was dissolved in phosphate-buffered saline(PBS) at pH 7.4 to a final concentration of 0.125%, 1% and 5% (w/v)along with a radioactivity of 0.625 uCi/25 ul.

B. Methods

New Zealand white female rabbits weighing 2.0-3.0 kg were treated as inExample 2B except that 25 ul of radioactive enkephalin solution (0.625uCi/26 ul) was used and blood samples collected for a total of 12 hours,rather than 6 hours. At the end of blood collection, the rabbits wereeuthanized as described above, the left eye enucleated and dissected,and tissues analyzed as described in Example 3B. The internal standardwas prepared by using the corresponding enkephalin with lowerradioactivity (0.0025 uCi/10 ul). The i.v. injection of enkephalin at 50ug (with 0.125 uCi/50 ul of radioactivity) was also carried out. Onemilliliter of blood sample was collected at 0, 2, 4, 6, 10, 20, 30, 60,90, 120, 150, 180, 240, and 300 minutes intervals. The blood sample wasreplaced by equal volume of heparinized normal saline. The radioactivityof blood samples was determined with a gamma counter. The internalstandard was prepared with corresponding radioactive enkephalin solutionwith a radioactivity of 0.0125 uCi/10 ul. The concentrations ofenkaphalin-equivalent in the blood were expressed in ng/ ml.

C. Results

All data were analyzed with Student's t-test for two values and analysisof variance for more than two values. Each value was expressed asmean±standard error of the mean. A p value of 0.05 or less wasconsidered significant.

When 25 ul of 0.125% enkephalin was instilled locally into the left eye,it entered systemic circulation rapidly to reach a plateau of 11.5 ng/mlin 3-4 hrs and remained in the plateau for the rest of experimentalperiod (8-9 hrs) (FIG. 17). With higher concentrations at 1% and 5%,similar absorption kinetics of enkephalin were observed except that theyreached higher plateaus of 72 ng/ml and 233 ng/ml, respectively (FIGS.17 and 18).

Intravenous administration of enkephalin (50 ug i.v.) resulted in rapiddecline of blood concentration with a T_(x) of less than 30 min andreached the lowest point at 22 ng/ml in 5 hrs (FIG. 17). These resultsindicate that local administration of enkephalin through eyes canmaintain a high blood concentration for a long period of time while theblood concentrations of enkephalin decline steadily with i.v. injection.

The residual amount of enkephalin remaining in the eye tissues wasminimal at best at 0.12-0.30% of total amount instilled into eyes (Table6). Most of the enkephalin remained in the cornea where the drug haddirect contact with the tissue. Although the total amount of enkephalinin the lens appeared high, it was because of the larger mass of thelens. The actual concentration of enkephalin in the lens was the lowestamong all tissues.

                                      TABLE 6                                     __________________________________________________________________________    The amount of enkephalin-equivalent remaining in eye tissues at the end       of 12 hr experiment                                                                  Residual amount of enkephalin remaining in tissues (ng/tissues)               0.125% (n = 5) 1% (n = 6)      5% (n = 5)                              Tissue Amount % of instilled                                                                        Amount  % of instilled                                                                        Amount   % of instilled                 __________________________________________________________________________    Cornea 44.01 ± 4.53                                                                      0.1408 ± 0.015                                                                     150.27 ± 37.06                                                                     0.0601 ± 0.015                                                                      533.29 ± 149.69                                                                    0.0427 ± 0.012              Iris    7.89 ± 0.94                                                                      0.0252 ± 0.003                                                                      29.19 ± 12.69                                                                     0.0117 ± 0.005                                                                      53.86 ± 13.16                                                                      0.0043 ± 0.001              Ciliary-Body                                                                          4.89 ± 0.88                                                                      0.0156 ± 0.003                                                                      27.11 ± 12.82                                                                     0.0108 ± 0.005                                                                      52.01 ± 22.86                                                                      0.0042 ± 0.002              Lens   26.05 ± 3.94                                                                      0.0834 ± 0.013                                                                     120.78 ± 25.60                                                                     0.0483 ± 0.010                                                                      597.33 ± 372.51                                                                    0.0478 ± 0.030              Retina  3.69 ± 1.39                                                                      0.0118 ± 0.004                                                                      39.72 ±  26.75                                                                    0.0155 ± 0.011                                                                      44.62 ± 22.41                                                                      0.0036 ± 0.002              Choroid                                                                               7.83 ± 2.67                                                                      0.0251 ± 0.0009                                                                     62.21 ± 39.73                                                                     0.0249 ± 0.016                                                                      137.29 ± 100.85                                                                    0.0110 ± 0.008              Total  94.36 ng                                                                             0.3019% 428.28 ng                                                                             0.1713% 1418.40 ng                                                                             0.1136%                        __________________________________________________________________________

EXAMPLE 5 Systemic Delivery Through the Eyes of Polypeptides WithMolecular Weights Between 300 and 3500

To test the effectiveness of the subject invention with respect topeptides of varying sizes, the following experiment was conducted asdescribed by Chiou, G. C. Y. and Chuang, C. Y. J., Ocular Pharm. (1988)4:165-177.

A. Materials

Thyrotrophin-releasing hormone (TRH), luteinizing hormone-releasinghormone (LH/RH) and glucagon were purchased from Sigma Chemical Company(St. Louis, MO). Thyrotropin-releasing hormone, [¹²⁵ I]-(spec, act.,2200 Ci/mmole); luteinizing hormone-releasing hormone, ¹²⁵ I]-(spec,act., 2200 Ci/mmole); and glucagon [¹²⁵ I]-(spec. act., 115 mCi/mg) werepurchased from DuPont NEN Research Products (Wilmington, DE). Peptidaseinhibitors, leucine-leucine, bestatin, and D,L-thiorphan were purchasedfrom Sigma Chemical Company.

All peptides were dissolved in phosphate-buffered saline (PBS) at pH 7.4to final concentration of 0.0025%, 1%, 5% (w/v) along with theradioactive peptides.

B. Methods

New Zealand white female rabbits weighing 2.0-3.0 kg were treated as inExample 2B. The internal standard was prepared by using the dilutedcorresponding peptide (0.0025 uCi/10 ul). An i.v. injection of peptidesat 0.625 ug (with 0.125 uCi/50 ul of radioactivity) was also carriedout. One milliliter of blood sample was collected at 0, 2, 4, 6, 10, 20,30, 60, 90, 150 180, 240 and 300 minute intervals. The blood volume wasreplaced by an equal volume of heparinized normal saline. Theradioactivity of blood samples was determined with a gamma counter. Theinternal standard was prepared with corresponding radioactive peptidesolution with a radioactivity of 0.0125 uCi/10 ul. The concentrations ofpeptides in the blood were expressed in ng/ml.

All data were analyzed with Student's t-test for two value and analysisof variance for more than two values. Each value was expressed asmean±standard error of the mean. A p value of 0.05 or less wasconsidered significant.

C. Results 1. Systemic absorption of topical TRH with a molecular weightof 300.

When 0.0025% of TRH was instilled in the eye, TRH reached a plateau of0.05 ng/ml in 60 min and remained at the plateau for at least 4 hrsthereafter (FIG. 19). In the clinics, 15 ug/70 kg of TRH is administeredintravenously. When the same dose was given intravenously to the rabbit,the blood concentration of TRH dropped quickly to the steady state of0.07 ng/ml in 2.5 hrs with the T_(x) of approximately 10 min (FIG. 19).With a higher dose at 1%, TRH reached a plateau of 26 ng/ml in 2 hrs andremained at the plateau for approximately 10 hrs thereafter (FIG. 20).At 5%, TRH peaked at 138 ng/ml in 60 min. Then the blood concentrationfell gradually to a steady state of 60 ng/ml in 9 hrs from the peak(FIG. 20). These results indicate that a therapeutic blood concentrationof TRH can be obtained with a low concentration of TRH (0.0025% orhigher) and the duration of action can last much longer than an i.v.injection dose. It also demonstrated that the systemic absorption of TRHthrough eyes is dose- and/or concentration-related (FIGS. 19 and 20).Addition of peptidase inhibitors (Leu-Leu, 4 mM; Bestatin, 60 uM; andD,L-Thiorphan, 0.6 uM) did not significantly enhance the systemicabsorption of TRH.

2. Systemic absorption of topical LH-RH with a molecular weight of 1200

To test whether a larger molecule could be absorbed effectively intosystemic circulation through the eyes, the experiment was repeated usingLHRH (FIG. 21). At 0.0025%, LHRH blood concentration increased graduallyover the 5 hr period to reach the final highest concentration of 0.13ng/ml. No peak or plateau was observed within 5 hrs after druginstillation (FIG. 21). When a therapeutic dose (15 ug/70 kg) of LHRHwas given intravenously, the blood concentration of LHRH fell rapidly to0.25-0.3 ng/ml in 30-180 min with a T_(x) of approximately 15 min. (FIG.21). The kinetics of LHRH absorption through the eyes at higherconcentrations (1% and 5%) was the same as 0.0025% solution except 1%solution reached 45 ng/ml in the blood at the end of 12 hrs whereas 5%solutions reached 95 ng/ml at the end of the same time period (FIG. 22).These results indicate that topical administration of LHRH to the eye ata concentration as low as 0.005% would be sufficient to reachtherapeutic effective blood concentration and this is superior over i.v.administration. A good dose-absorption relationship was alsodemonstrated in this case (FIGS. 21 and 22). Addition of a peptidaseinhibitor (Leu-Leu, 5 mM) did increase the systemic absorption of LHRHsignificantly (FIG. 23).

3. Systemic absorption of topical glucagon with a molecular weight of3500

The systemic absorption kinetics of glucagon through eyes was verysimilar to TRH. At 0.0025%, glucagon reached a plateau of 0.25 ng/ml in2.5 hrs and persisted in that concentration for the rest of theexperimental period (at least 3.5 hrs) (FIG. 24). When 0.625 ug ofglucagon was given intravenously, the blood concentration droppedprecipitously with a T_(x) of approximately 10 min and remained low at0.27-0.3 ng/ml for 5 hrs (FIG. 24). At 1%, glucagon reached the plateauof 60 ng/ml in the blood in 3 hrs and remained at plateau for the next 9hrs. At 5%, topical glucagon reached 155 ng/ml in the blood in 4 hrs andpersisted at the same level for 8 hrs thereafter (FIG. 25). Theseresults again indicate that topical instillation of peptides results intheir effective absorption into systemic circulation. There was also agood dose-absorption relationship with glucagon (FIGS. 32 and 33).Addition of peptidase inhibitor (Leu-Leu, 5 mM) did not increase thesystemic absorption of glucagon.

4. Absorption of topical peptides into the eyes

The amount of peptides absorbed into eyes at 12 hrs after druginstillation is presented in Tables 7-9. The amount of peptides in theeye tissues was very low except cornea where the peptides had directcontact when applied.

                                      TABLE 7                                     __________________________________________________________________________    The amount of TRH remaining in eye tissues at the end of 12 hr                experiment                                                                           Residual amount of TRH remaining in tissue (ng/tissue)                        1% TRH (n = 5)  5% TRH (n = 5)                                         Tissue Amount  % of instilled                                                                        Amount  % of instilled                                 __________________________________________________________________________    Cornea  60.40 ± 33.49                                                                     0.0242 ± 0.0134                                                                    334.82 ± 93.38                                                                     0.0268 ± 0.0075                             Iris    8.85 ± 2.93                                                                       0.0035 ± 0.0012                                                                     78.67 ± 22.95                                                                     0.0063 ± 0.0018                             Ciliary-Body                                                                          8.49 ± 2.33                                                                       0.0034 ± 0.0009                                                                     45.72 ± 14.02                                                                     0.0037 ± 0.0011                             Lens    28.45 ± 7.15                                                                      0.0114 ± 0.0029                                                                    109.80 ± 27.87                                                                     0.0088 ± 0.0022                             Retina  4.91 ± 2.40                                                                       0.0020 ± 0.0010                                                                     17.96 ± 5.41                                                                      0.0014 ± 0.0004                             Choroid                                                                               6.60 ± 2.75                                                                       0.0026 ± 0.0011                                                                     24.20 ± 4.81                                                                      0.0019 ± 0.0004                             TOTAL  117.70 ng                                                                             0.0471% 611.17 ng                                                                             0.0489%                                        __________________________________________________________________________

                                      TABLE 8                                     __________________________________________________________________________    The amount of LH-RH remaining in eye tissues at the end of 12 hr              experiment                                                                           Residual amount of LH-RH remaining in tissue (ng/tissue)                      1% LH-RH (n = 5)                                                                              5% LH-RH (n = 6)                                       Tissue Amount  % of instilled                                                                        Amount   % of instilled                                __________________________________________________________________________    Cornea 360.89 ± 97.08                                                                     0.1444 ± 0.0388                                                                     812.37 ± 257.35                                                                    0.0650 ± 0.0206                            Iris    61.04 ± 16.66                                                                     0.0244 ± 0.0067                                                                     97.97 ± 33.55                                                                      0.0078 ± 0.0027                            Ciliary-Body                                                                          38.81 ± 13.28                                                                     0.0155 ± 0.0053                                                                     79.93 ± 29.13                                                                      0.0064 ± 0.0023                            Lens   157.48 ± 43.43                                                                     0.0630 ± 0.0174                                                                     263.79 ± 92.59                                                                     0.0211 ± 0.0074                            Retina  16.70 ± 4.68                                                                      0.0067 ± 0.0019                                                                     70.45 ± 26.72                                                                      0.0056 ± 0.0021                            Choroid                                                                               53.92 ± 20.19                                                                     0.0216 ± 0.0081                                                                     279.96 ± 145.17                                                                    0.0224 ± 0.0116                            TOTAL  688.84 ng                                                                             0.2756% 1604.47 ng                                                                             0.1283%                                       __________________________________________________________________________

                                      TABLE 9                                     __________________________________________________________________________    The amount of glucagon remaining in eye tissues at the end of 12 hr           experiment                                                                           Residual amount of glucagon remaining in tissues (ng/tissue)                  1% glucagon (n = 6)                                                                           5% glucagon (n = 5)                                    Tissue Amount  % of instilled                                                                        Amount   % of instilled                                __________________________________________________________________________    Cornea 204.41 ± 106.78                                                                    0.0818 ± 0.0427                                                                    1100.16 ± 463.06                                                                    0.0880 ± 0.0370                            Iris    16.82 ± 10.74                                                                     0.0067 ± 0.0043                                                                     122.67 ± 40.04                                                                     0.0098 ± 0.0032                            Ciliary-Body                                                                          12.61 ± 6.89                                                                      0.0050 ± 0.0028                                                                     77.28 ± 24.03                                                                      0.0062 ± 0.0019                            Lens   112.31 ± 57.92                                                                     0.0449 ± 0.0232                                                                     693.48 ± 318.47                                                                    0.0555 ± 0.0255                            Retina  15.93 ± 8.12                                                                      0.0064 ± 0.0033                                                                     84.71 ± 31.37                                                                      0.0068 ± 0.0025                            Choroid                                                                               14.01 ± 7.56                                                                      0.0056 ± 0.0030                                                                     132.27 ± 40.90                                                                     0.0106 ± 0.0033                            TOTAL  376.09 ng                                                                             0.1504% 2210.57 ng                                                                             0.1769%                                       __________________________________________________________________________

These results, along with the previous experimental data, show thatpolypeptides of varying molecular weights find use with the presentinvention. Specifically, the efficacy of ocular administration ofinsulin (molecular weight 6000), glucagon (molecular weight 3500),enkephalin (molecular weight 600), TRH (molecular weight 300), and LH-RH(molecular weight 1200) has been demonstrated. Thus, it is expected thatunder the proper conditions, and with the use of permeation enhancers toaid absorption of the larger peptides, almost any peptide drug could beadministered using the subject invention.

Thus, compositions and methods for the systemic delivery of polypeptidesthrough the eyes have been disclosed. Although the subject invention hasbeen described with reference to several preferred embodiments, otherembodiments will readily occur to those of skill in the art. Thus, thescope of the present invention is defined by the following claimswithout limitation to the foregoing examples.

I claim:
 1. A pharmaceutical composition for systemic delivery by ocularadministration and absorption in the nasolacrimal duct comprising:asystemically active polypeptide in a pharmaceutically acceptablevehicle, the polypeptide being present at a concentration such that thetonicity of the composition is equivalent to the tonicity of 0.5% to1.8% sodium chloride solution; and a permeation-enhancing agent toenhance absorption of the polypeptide into the systemic circulation viathe mucous membrane of the conjunctiva and nasolacrimal system.
 2. Thecomposition of claim 1 wherein the permeation-enhancing agent is atleast one agent selected from the group consisting of polyoxyethylene,polyoxyethylene ethers of fatty acids, sorbitan monooleate, sorbitanmonolaurate, polyoxyethylene monolaurate, polyoxyethylene sorbitanmonolaurate, fusidic acid and derivatives thereof, EDTA, disodium EDTA,cholic acid, deoxycholic acid, glycocholic acid, glycodeoxycholic acid,taurocholic acid, taurodeoxycholic acid, sodium cholate, sodiumglycocholate, glycocholate, sodium deoxycholate, sodium taurocholate,sodium glycodeoxycholate, sodium taurodeoxycholate, chenodeoxycholicacid, urosdeoxycholic acid, saponins, glycyrrhizic acid, ammoniumglycyrrhizide, decamethonium, decamethonium bromide, anddodecyltrimethylammonium bromide.
 3. The composition of claim 1 whereinthe polypeptide is at least one polypeptide selected from the groupconsisting of insulin, beta-lipotropin, gammalipotropin, glucagon,leu-enkephalin, met-enkephalin, dynorphin A, dynorphin B,alpha-neoendorphin, betaneoendorphin, thyrotrophin releasing hormone,luteinizing hormone releasing hormone, oxytocin, vasopressin, lypressin,calcitonin, ACTH, growth, hormone releasing factor, gonadotropinreleasing hormone, somatotropin, somatostatin, secretin, concanavalin,ribonuclease, and lysozyme.
 4. The composition of claim 1 wherein theconcentration of polypeptide therein is less than 10% w/v.